The specific aim of this program is to design and synthesize improved active site directed inhibitors of cruzain, the major cysteine protease of Trypanosoma cruzi. Peptide mimetics and non-peptidic inhibitors will be designed with the aid of insights deriving from: (i) The X-ray structure recently solved by Fletterick and McGrath, which has a covalently bound Z-Phe-Ala-FMK inhibitor at the active site: (ii) Knowledge of the structural requirements of the dipeptide fluoromethyl ketones (FMK) that McKerrow and coworkers have demonstrated to be potent, irreversible inhibitors of purified cruzain, and also to be active in vivo against Trypanosoma cruzi; and, (iii) Structural information deriving from the DOCK-generated, non- peptidic lead inhibitor structures deriving from the work of Cohen and Ring. In addition, a new class of cysteine protease inhibitors based on the E- 64 motif will be developed. Specifically, inhibitors incorporating epoxypropionyl ketone (EPK) substructures will be synthesized and evaluated by our parasitology and biochemistry collaborators, McKerrow and Engel, at UC San Francisco. Further modifications and optimization of the initial EPK inhibitors will be performed if promising activity as cruzain inhibitors is observed. The focus of these efforts is to identify modifications of the lead inhibitor structures already identified (see Background and Significance Section) so as to enhance their use in vivo. Factors such as absorption from the oral route, eliminating functional groups that may contribute to toxic side reactions, and enhancing the activity and specificity of the inhibitors against the targeted protease, cruzain, will be evaluated in iterative cycles involving our synthesis group, the computer modeling group (Cohen), the protein structure groups (McGrath, Fletterick and Craik), and the parasitology and biochemistry components of this program (McKerrow and Engel). These combined efforts will lead to the design and synthesis of ever more specific and potent cruzain inhibitors.